US6994959B1 - G-rich oligo aptamers and methods of modulating an immune response - Google Patents
G-rich oligo aptamers and methods of modulating an immune response Download PDFInfo
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- US6994959B1 US6994959B1 US09/331,204 US33120499A US6994959B1 US 6994959 B1 US6994959 B1 US 6994959B1 US 33120499 A US33120499 A US 33120499A US 6994959 B1 US6994959 B1 US 6994959B1
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2310/00—Structure or type of the nucleic acid
- C12N2310/30—Chemical structure
- C12N2310/31—Chemical structure of the backbone
- C12N2310/315—Phosphorothioates
Definitions
- the field of the invention is immunology.
- T-cell activation The pathogenesis and exacerbation of many prevalent T-cell mediated diseases result from an inappropriate immune response driven by abnormal T-cell activation.
- a number of other diseases are thought to be caused by aberrant T-cell activation including Type I (insulin-dependent) diabetes mellitus, thyroiditis, sarcoidosis, multiple sclerosis, autoimmune uveitis, rheumatoid arthritis, systemic lupus erythematosus, inflammatory bowel disease (Crohn's and ulcerative colitis) and aplastic anemia.
- a variety of syndromes including septic shock and tumor-induced cachexia may involve T-cell activation and augmented production of potentially toxic levels of lymphokines.
- Normal T-cell activation also mediates the rejection of transplanted cells and organs by providing the neccessary signals for the effective destruction of the “foreign” donor tissue.
- T-lymphocytes leading to T-cell proliferation and gene expression and secretion of specific immunomodulatory cytokines requires two independent signals.
- the first signal involves the recognition, by specific T-cell receptor/CD3 complex, of antigen presented by major histocompatibility complex molecules on the surface of antigen-presenting cells (APCs).
- APCs antigen-presenting cells
- Antigen-nonspecific intercellular interactions between T-cells and APCs provide the second signal which serves to regulate T-cell responses to antigen.
- These secondary or costimulatory signals determine the magnitude of a T-cell response to antigen. Costimulated cells react by increasing the levels of specific cytokine gene transcription and by stabilizing selected mRNAs.
- T-cell activation in the absence of costimulation results in an aborted or anergic T-cell response.
- One key costimulatory signal is provided by interaction of the T-cell surface receptor CD28 with B7-related molecules on APC (Linsley and Ledbetter (1993) Annu Rev Immunol 11: 191–212).
- CD28 is constitutively expressed on 95% of CD4 + T-cells (which provide helper functions for B-cell antibody production) and 50% of CD8 + T-cells (which have cytotoxic functions) (Yamada et al (1985) Eur J Immunol 15: 1164–1168).
- IL-2 interleukin-2
- IFN ⁇ interferon-gamma
- IL-8 interleukin-8
- cytokines have been shown to be regulated by the CD28 pathway of T-cell activation (Fraser et al (1991) Science 251: 313–316, Seder et al (1994) J Exp Med 179: 299–304, Wechsler et al (1994) J Immunol 153: 2515–2523).
- IL-2, IFN ⁇ and IL-8 are essential in promoting a wide range of immune responses and have been shown to be overexpressed in many T-cell mediated disease states.
- activated lesional T-cells predominantly release Th1 cytokines such as IL-2 and IFN ⁇ (Schlaak et al (1994) J Invest Derm 102: 145–149). These secreted cytokines induce normal keratinocytes to express the same phenotype (HLA DR + /ICAM-1 + ) as found in psoriasis lesions (Baadsgaard et al (1990) J Invest Derm 95: 275–282).
- Th1 cytokines such as IL-2 and IFN ⁇
- IL-8 by virtue of its in vitro and in vivo proinflammatory properties and because it is secreted in large amounts by both activated T-cells and keratinocytes from psoriatic lesions, is considered a major contributor to the pathologic changes seen in psoriatic skin such as keratinocyte hyperproliferation.
- BB1 one of the B7 family of receptors (the natural ligands for CD28 found on activated APC), BB1 has been shown to be expressed in psoriatic but not unaffected skin keratinocytes (Nickoloff et al (1993) Am J Pathology 142: 1029–1040) underscoring the importance of T-cell activation in pathogenesis of the disease.
- CD28 was expressed in high levels in the majority of dermal and epidermal CD3 + T-cells, but in normal skin and basal cell carcinoma (a non T-cell mediated skin disease), CD28 was expressed only in perivascular T-cells.
- B7 expression was found on dermal dendritic cells, dermal APCs and on keratinocytes, but not in normal skin and basal cell carcinoma (Simon et al (1994) J Invest Derm 103: 539–543). Therefore this suggests that the CD28/B7 pathway is an important mediator of T-cell-mediated skin diseases.
- T-cell activation associated with certain autoimmune diseases caused by the loss of self-tolerance is predominantly characterized by the presence of CD28 + T-cells and expression of its ligand, B7 on activated professional APCs (monocyte, macrophage or dendritic cells).
- autoimmune diseases do not prevent T-cell activation; the effector step in the autoreactive immune responses to self-antigen.
- Drugs such as steroids and non-steroid anti-inflammatory drugs (NSAIDS)
- NSAIDS non-steroid anti-inflammatory drugs
- steroids can have side effects such as inducing osteoporosis, organ toxicity and diabetes, and can accelerate the cartilage degeneration process and cause so-called post-injection flares for up to 2 to 8 hours.
- NSAIDS can have gastrointestinal side effects and increase the risk of agranulocytosis and iatrogenic hepatitis.
- Immunosuppressive drugs are also used as another form of therapy, especially in advanced disease stages. However, these drugs suppress the entire immune system and often treatment has severe side effects including hypertension and nephrotoxicity. Also established immunosuppressants such as cyclosporin and FK506 cannot inhibit the CD28-dependent T-cell activation pathway (June et al (1987) Mol Cell Biol 7: 4472–4481).
- T-cell activation molecules include synthetic peptides, monoclonal antibodies and soluble forms of T-cell activation molecules. To date competitive synthetic peptides to T-cell activation molecules such as CD28, CD40L and the CAM family of adhesion molecules have not been identified. Monoclonal antibodies (mAb) have been shown to have possible therapeutic effect in such T-cell mediated diseases such as psoriasis (anti-CD4 (Prinz et al (1994) Lancet 338: 320–321)) and immununosuppression of normal T-cell activation in allografts (anti-VCAM-1 and VLA-4 (Isobe et al (1994) J Immunol 153: 5810–5818)).
- psoriasis anti-CD4 (Prinz et al (1994) Lancet 338: 320–321)
- immununosuppression of normal T-cell activation in allografts anti-VCAM-1 and VLA-4
- CTLA-4Ig has been shown to specifically block normal T-cell activation by preventing rejection of xenogeneic (Lenschow et al (1992) Science 257: 789–792) and allogeneic (Turka et al (1992) Proc Nat Acad Sci USA 89: 11102–11105) cardiac allografts in rats and have therapeutic effect on aberrant T-cell activation such as found in rat autoimmune glomerulonephritis (Nishikawa et al (1994) Eur J Immunol 24: 1249–1254). Soluble CTLA-4Ig however suffers from similar limitations as monoclonal antibodies in addition to the expense of their production. Also the true function of this CD28-like molecule is not known therefore this needs to be fully determined before any therapeutic benefit can be evaluated.
- Inhibition of the cell-surface expression of CD28 leads to prolonged unresponsiveness or deletion of activated T-cells. Inactivation prevents T-cell proliferation and arrest of T-cell-specific production of specific immunoregulatory cytokines such as interleukin-2, interferon-gamma and interleukin-8.
- oligonucleotides By hybridizing oligodeoxy-ribonucleotides or oligoribonucleotides to DNA or RNA sequences within the CD28 gene or promoter region (See PCT/US96/01507, filed Aug. 30, 1996). Oligonucleotides avoid many of the pitfalls of current agents used to block the effects of normal and abnormal T-cell activation. However, these oligos designed for antisense strategies are susceptible to degradation by intracellular nucleases or nucleases present in the extracellular milieu.
- DNA or RNA
- RNA DNA
- G guanosine
- C cytosine
- A adenosine
- T thymidine
- transcription factors are a class of proteins which regulate genes by primarily binding to specific regulatory sequences in the 5′ upstream promoter region of those genes. This interaction leads to initiation of transcription.
- Certain transcription factors such as Sp1, AP2, AP-1, EGR-1 and NF ⁇ B are critical in the activation of T and B lymphocytes (Skerka et al J Biol Chem 270: 22500–22506, Jung et al (1995) Ann N Y Acad Sci 766: 245–252).
- the present invention provides aptamers having a length of between about 12 and 22 nucleic acid units, inclusive, and a sequence which includes at least two G-rich regions selected from the group consisting of GGnG, GGGG, GnGG, nGGG and GGGn, where G is guanidine and n is any nucleotide.
- the oligonucleotides are designed to bind to specific regulatory proteins such as Sp1 and Sp1-related proteins and act to compete with the binding of these transcription factors to the promoter region of the genes which are under their control. This serves to modulate gene expression by preventing transcription of the gene.
- the aptamer oligonucleotides are able to inhibit the function of RNA or DNA, either its translation into protein, its translocation into the cytoplasm or any other activity neccessary to its overall biological function. The failure of the RNA or DNA to perform all or part of its function results in failure of a portion of the genome controlling T-cell activation to be properly expressed, thus modulating said metabolism.
- CD28 protein is particularly useful for this approach. Inhibition of CD28 and CD28-related gene expression is expected to be useful for the treatment of psoriasis and other skin diseases, syndromes with aberrant T-cell activation, autoimmune disorders and allograft rejection.
- Methods of modulating T-cell activation comprising contacting a patient with an oligonucleotide which competes with the DNA-binding site of a regulatory protein such as to inhibit expression of a regulated protein known to be capable of modulating T-cell activation.
- Oligonucleotides which bind to proteins such as Sp1 and Sp1-related proteins which regulate transcription of CD28 and CD28-related genes are preferred.
- aptamers are administered to provide therapies for diseases which involve aberrant T-cell activation such as psoriasis, AIDS-exacerbated psoriasis and other skin diseases, Type I (insulin-dependent) diabetes mellitus, thyroiditis, sarcoidosis, multiple sclerosis, autoimmune uveitis, rheumatoid arthritis, systemic lupus erythematosus, inflammatory bowel disease (Crohn's and ulcerative colitis), septic shock, tumor-induced cachexia and aplastic anemia and to regulate normal T-cell activation such as in allograft rejection.
- This can be achieved by perturbation in the synthesis and expression of T-cell activation molecules including CD28 and CD28-related molecules.
- aptamers are provided which are capable of binding specific regulatory proteins such as Sp1 and Sp 1-related proteins and thus inhibit transcription of genes such as CD28 and CD28-related proteins which a) are normally regulated by these proteins and b) can modulate T cell responses.
- FIGS. 1A and 1B are graphical representations of the in vitro stability of 32 P-labeled phosphorothioate, ICN 16064 (Seq #4), in extracellular fluid and in Jurkat cells, respectively.
- FIGS. 1C and 1D are graphical representations of the in vitro stability of 32 P-labeled phosphorothioate, ICN 16214 (Seq #21), in extracellular fluid and in Jurkat cells, respectively.
- FIGS. 1E and 1F are graphical representations of time-dependent degradation (0–96 h) of each oligonucleotide ICN 16064 (Seq #4) and ICN 16214 (Seq. #21), (2000 cpm) as assessed by electrophoresis on a 20% polyacrylamide denaturing gel followed by visualization using a Phosphorlmager.
- the percentage of intact full length 32 P-RT03S ( ⁇ ) and 32 P-RTCO6S ( ⁇ ) remaining at each time point, relative to t 0, was determined in eluates from 10000 cpm of extracellular ( FIG. 1E ) and cell ( FIG. 1F ) applied through Nickspin columns (Pharmacia).
- Molecular weight standards (Std), 32 P-dNTP (N) and free 32 P-orthophosphate (P) were simultaneously analysed.
- FIG. 2 is a graphical representation of a gel shift analysis which demonstrates that oligonucleotides containing a G-rich 12mer sequence motif (lane 5 and 11) give a distinct band A which differs in electrophoretic shift to band B observed with other phosphorothioate oligonucleotides following incubation with HeLa nuclear extract.
- Band C is 32P-oligo alone.
- FIG. 3 is a graphical representation of chloramphenicol acetyltransferase (CAT) expression following the transfection of Jurkat cells with plasmid vectors containing a 226 bp insert from the CD28 promoter region (residues ⁇ 197 to +28) (28b) or a mutant with a substitution at residues ⁇ 51 to ⁇ 22 with Seq#3 from Table 1, (28h-1) upstream of the CAT reporter gene, and following treatment with and without the phosphorothioate oligonucleotides, ICN16064 and ICN 16481.
- CAT chloramphenicol acetyltransferase
- FIG. 4 is a graphical representation of a gel supershift assay showing that the binding of Sp1 to 28b, the upstream region ⁇ 197 to +28 of the CD28 gene is specific.
- FIG. 5 is a graphical representation of the binding of Sp1 to the 32 P-labeled double stranded oligo 28b (which is derived from the parent 28b—Seq #1 Table 1) and the competition binding of cold double stranded oligo 28b and the aptameric oligos FIG. 1A and 16481.
- oligonucleotides that specifically bind to the DNA-binding site of regulatory proteins such as Sp1 and Sp1-related proteins will prevent the binding of the regulatory protein with specific double-stranded region of DNA in the promoter region the gene of interest.
- the competive binding by the aptamer would hinder transcription of the gene and thus inhibit the flow of genetic information from DNA to protein.
- the properties of oligonucleotides which make them specific for their target also make them versatile. Because oligonucleotides are long chains of four monomeric units they may be readily synthesized for any target RNA sequence.
- Oligonucleotide-mediated inhibition of gene expression has been demonstrated in many model and in vitro systems and has therapeutic potential as a new strategy for treating many human diseases (Uhlmann and Peyman (1990) Chem Rev 90: 544–584, Zon and Stec (1991) Oligonucleotides and analogues—A Practical Approach: 87–108, Miller et al (1981) Biochem 20: 1874–1880, Orson et al (1991) Nucleic Acid Res 19: 3435–3441, Helene and Toulme (1990) Biochem Biophys Acta 1049: 99–125, Thierry and Dritschilo (1992) Nucleic Acid Res 20: 5691–5698).
- oligonucleotides and analogues A Practical Approach: 87–108 and phosphorothioate-3′hydroxypropylamine (Tam et al (1994) Nucleic Acid Res 22: 977–986), which exhibit enhanced cell uptake, it is now possible to consider the use of oligonucleotides as a novel form of therapeutics.
- Aptameric oligonucleotides targeting regulatory protein binding sites represent an alternative class of nucleic acid-based compounds and they offer an ideal solution to the problems encountered in prior art approaches.
- Oligonucleotides are small molecules and therefore do not encounter the same steric problems as large molecule inhibitors.
- Targets comtemplated herein include molecules which can be regulated by transcription factors which play an essential role in initiating or maintaining an immune response. These include the costimulatory molecules such as CD28 and cytokines such as IL-2, GM-CSF and IFN ⁇ .
- oligonucleotides may be formulated in a pharmaceutical composition, which may include carriers, thickeners, diluents, buffers, preservatives, surface active agents, liposomes or lipid formulations and the like in addition to the oligonucleotide.
- Pharmaceutical compositions may also include one or more active ingredients such as antimicrobial agents, anti-inflammatory agents, anesthetics, and the like in addition to oligonucleotide.
- the pharmaceutical composition may be administered in a number of ways depending on whether local or systemic treatment is desired, and on the area to be treated. Adminstration may be topically (including opthalmically, vaginally, rectally, intranasally), orally, by inhalation, or parenterally, for example by intravenous drip, subcutaneous, intraperitoneal or intramuscular injection.
- Formulations for topical administration may include ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders.
- Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be neccessary or desirable.
- Coated condoms or gloves may also be useful.
- compositions for oral administration include powders or granules, suspensions or solutions in water or non-aqueos media, capsules, sachets or tablets. Thickeners, flavorings, diluents, emulsifiers, dispersing aids or binders may be desirable.
- Formulations for parenteral administration may include sterile aqueous solutions which may contain buffers, liposomes diluents and other suitable additives.
- Dosing is dependent on severity and responsiveness of the condition to be treated, but will normally be one or more doses per day, with course of treatment lasting from several days to several months or until a cure is effected or a diminution of disease state is achieved. Persons of ordinary skill can easily determine optimum dosages, dosing methodologies and repetition rates.
- the aptamers are to be administered intravenously in a dose of 5 mg/kg once per day.
- the aptamers are to be administered in a 1–5% solution once per day.
- the aptamers are to be administered in an aerosolized dose of 5 mg once per day.
- oligonucleotide refers to an oligomer or polymer of ribonucleic acid or deoxyribonucleic acid. This term includes oligomers consisting of naturally occurring bases, sugars and intersugar (backbone) linkages as well as oligomers having non-naturally occurring portions which function similarly. Such modified or substituted oligonucleotides are often preferred over native forms because of properties such as, for example, enhanced cellular uptake and increased stability in the presence of nucleases.
- the oligonucleotides in accordance with this invention preferably comprise from about 3 to about 50 nucleic acid base units. It is more preferred that such oligonucleotides comprise from about 8 and 30 nucleic acid base units, and still more preferred to have from about 12 and 22 nucleic acid base units.
- a nucleic acid base unit is a base-sugar combination suitably bound to an adjacent nucleic acid base unit through phosphodiester or the other bonds.
- oligonucleotides used in accordance with this invention may be conveniently and routinely made through the well-known technique of solid phase synthesis.
- messenger RNA identified by the open reading frames (ORFs) of the DNA from which they are transcribed includes not only the information from the ORFs of the DNA, but also associated ribonucleotides which form regions known to such persons as the 5′-untranslated, the 3′-untranslated region and intervening sequence ribonucleotides.
- ORFs open reading frames
- oligonucleotides may be formulated in accordance with this invention which are targeted wholly or in part to these associated ribonucleotides as well as to the informational ribonucleotides.
- the aptameric oligonucleotide interacts with the DNA-binding site of a regulatory protein such as Sp1 and Sp1-related proteins, and in doing so interrupt the expression of a gene encoding a protein involved in T-cell activation.
- said proteins to be regulated are CD28 and all homologues of the CD28 molecule.
- preferred sequence segments which may be useful in whole or in part are:
- FIG. 1A GGG GAG GAG GGG CTG GAA ICN 16481 GGG GTG GTG GGG ICN 16525 TTG GAG GGG GAG GAG GGG ICN 16475 TTG GAG GGG GAG GTG GGG ICN 16479 GGG TTG GAG GGG GTG GTG GGG ICN 16065 While the illustrated sequences are believed to be accurate, the present invention is directed to the correct sequences should errors be found. Oligonucleotides useful in the invention comprise one of these sequences, or part thereof.
- any of these oligonucleotides as set forth above, or any of the similar oligonucleotides which persons of ordinary skill in the art can prepare from knowledge of the preferred oligonucleotide targets for the modulation of the synthesis of T-cell activation molecules including CD28 and CD28-related molecules.
- the inhibition or modulation of production of the CD28 and/or CD28 homologues are expected to have significant therapeutic benefits in the treatment of disease.
- an assay or series of assays is required.
- Oligodeoxynucleotides were synthesized on an automated DNA synthesizer (Applied Biosystems model 394) using standard phosphoramidite chemistry. ⁇ -cyanoethylphosphoramidites, synthesis reagents and CPG polystyrene columns were purchased from Applied Biosystems (ABI, Foster City, Calif.). 3′-Amino-Modifier C3 CPG columns were purchased from Glen Research (Sterling, Va.).
- phosphorothioate oligonucleotides For phosphorothioate oligonucleotides, the standard oxidation bottle was replaced with tetraethylthiuram disulfide/acetonitrile, and the standard ABI phosphorothioate program was used for the stepwise addition of phosphorothioate linkages. After cleavage from the controlled pore glass column, the protecting groups were removed by treating the oligonucleotides with concentrated ammonium hydroxide at 55° C. for 8 hours. The oligonucleotides were purified by HPLC using a reverse phase semiprep C8 column (ABI).
- ABSI reverse phase semiprep C8 column
- PBMCs Peripheral blood mononuclear cells
- RPMI-AP5 RPMI-1640 medium (ICN, Costa Mesa, Calif.) containing 20 mM HEPES buffer, pH 7.4, 5% autologous plasma, 1% L-glutamine, 1% penicillin/streptomycin and 0.05% 2-mercaptoethanol) to remove any contaminating adherent cells.
- RPMI-1640 medium ICN, Costa Mesa, Calif.
- autologous plasma 1% L-glutamine
- penicillin/streptomycin 0.05% 2-mercaptoethanol
- the T-cell lymphoma cell line, Jurkat E6-1 (CD28 + /CD4 + ) cells (152-TIB) were maintained in RPMI-10 (RPMI-1640 medium containing 20 mM HEPES buffer, pH 7.4, 10% fetal calf serum (FCS) (Hyclone, Logan, Utah), 1% L-glutamine and 1% penicillin/streptomycin).
- RPMI-10 RPMI-1640 medium containing 20 mM HEPES buffer, pH 7.4, 10% fetal calf serum (FCS) (Hyclone, Logan, Utah), 1% L-glutamine and 1% penicillin/streptomycin).
- Anti-CD3/PMA-activated T-cells were treated with 1–20 ⁇ M CD28-specific and control oligonucleotides immediately following activation and re-treated 24 h later. T-cells from one duplicate plate was used for immunofluorescence analysis and the 1 A used for cytokine studies and the second plate was used for T-cell proliferation analysis.
- 150 ⁇ l cell supernatant from the first duplicate microplate was transferred to another microplate for analysis of cell-derived cytokine production.
- the remaining cells were washed twice with isotonic saline solution, pH 7.4 (Becton Dickinson, Mansfield, Mass.) and resuspended in 50 ⁇ l isotonic saline solution and split into two samples.
- One sample aliquot was co-stained with either PE-CD28/FITC-CD4 mAb and non-specific fluorescence was assessed by staining the second aliquot with PE/FITC-labeled isotype-matched control monoclonal antibody.
- control untreated and oligonucleotide-treated cells were determined in each batch of all oligonucleotides in multiple donors by staining with the vital dye, propidium iodide (5 ⁇ g/ml final concentration).
- the percentage of live cells which excluded propidium iodide was determined by flow cytometry and was >90% (range 90–99%) following treatment with all batches of all oligonucleotides at a dose range of 1–20 ⁇ M.
- IL-2 interleukin-2
- Test oligonucleotides were labeled at the 5′ end with [ ⁇ -32P]-ATP (ICN, Costa Mesa, Calif.) using T4 polynucleotide kinase as per manufacturers protocol (Gibco BRL, Gaithersburg, Md.). 10 ⁇ g of HeLa cell nuclear extract (Promega) was incubated with approximately 80,000 cpm of labeled oligonucleotide for 20 min at room temperature.
- the binding reaction mixtures contained 10 mM Tris-HCl (pH 7.5), 50 mM NaCl, 0.5 mM DTT, 0.5 mM EDTA, 1 mM MgCl 2 , 4% glycerol and 0.5 ⁇ g of poly(dI.dC).
- DNA-protein complexes were resolved by electrophoresis through a 4% polyacrylamide gel containing 0.5 ⁇ TBE buffer (50 mM Tris, 45 mM boric acid, 0.5 mM EDTA) for approximately 3 hr at 100 V. The gel was dried and autoradiographed using Phosphorlmager (Biorad, Richmond, Calif.).
- the cDNAs (about 300 base pairs) used in the protein-DNA binding in DNase footprint assays and gel shift assays were isolated from plasmids pCAT3e 28b, pCAT3e 28h or pCAT3e 28h-1. 60 ⁇ g of each plasmid were digested with BglII, some being put first on agarose gel to check for linearity, the rest then phenol/sevag extracted, ethanol precipitated and then resuspended in water and digested with SacI. Again, a small portion was put on the gel to check if it's cut. (2 bands should appear now: a 4 kb band and a 300 b.p.
- DNA pellet was resuspended in a small volume of water (62 ⁇ l), 1 ⁇ l of 20 U/ ⁇ l alkaline phosphatase (Boehringer Mannheim, Indianapolis, Ind.) and 7 ⁇ l of the 10 ⁇ reaction buffer were added. After incubating the reaction mixture at 37° C. for 1 hour, 7 ⁇ l of pH 8.0, 0.2M EGTA was added and the whole tube was heated at 65° C. for 10 min. The whole 77 ⁇ l of the dephosphorylated DNA were put on 1% agarose gel to purify the 300 b.p.
- 4% non-denaturing polyacrylamide gel solution in 0.5 ⁇ TBE was prepared according to the Promega Gel Shift Assay Systems technical bulletin (4% acrylamide, 0.05% bisacrylamide, 2.5% glycerol, 0.5 ⁇ TBE).
- a stock of 250 ml of the above gel solution was prepared, filtered and kept at 4° C.
- 12.5 ⁇ l of TEMED and 187.5 ⁇ l of 10% ammonium persulfate was added to every 25 ml of the stock 4% gel solution and poured into 16.5 cm ⁇ 16.5 cm ⁇ 0.75 mm glass plates. Gels were always allowed to polymerize overnight for optimal results.
- the gel was pre-run in 0.5 ⁇ TBE buffer for 30 min at 100V before loading the samples.
- the method used here was from “Antiparallel polypurine phosphorothioate oligonucleotides form stable triplexes with the rat ⁇ 1(I) collagen gene promoter and inhibit transcription in cultured rat fibroblasts” by Jacob Joseph et al. in Nucleic Acids Research, 1997, Vol. 25, No. 11, 2182–2188. Equal amounts of complementary single strands were heated at 80° C. for 5 min in 0.25M NaCl, followed by slow cooling to room temp.
- Annealed double-stranded oligonucleotides were purified by electrophoresis on a 6% non-denaturing (29:1) polyacrylamide gel, later cut out, “crushed and soaked,” ethanol precipitated using the methods described in “Molecular cloning, a laboratory manual” by Sambrook, Fritsch & Maniatis. About 20 ng of d.s. oligo were used in each labeling reaction.
- Proteins (nuclear extract or purified transcriptional factor) were incubated with 1 ⁇ gel shift buffer (Promega, Madison, Wis.) at room temp for 5–10 min before kinased DNA was added and incubated for another 20–30 min at room temp. Samples were then loaded on the pre-run 4% non-denaturing gel. After about 3–4 hours run at 100V in 0.5 ⁇ TBE, gel was dried on 2 pieces of Whatman papers and exposed in a phosphor-imager overnight.
- 1 ⁇ gel shift buffer Promega, Madison, Wis.
- Antibody to Sp1 (clone 1C6, Santa Cruz Biotechnologies, Santa Cruz, Calif.) was pre-incubated with purified Sp1 (Promega) for 1 hour before the 32 P-labeled cDNA or oligo was added.
- CD28 upstream cDNA ( ⁇ 197 to +28) was produced by RT PCR using Jurkat total RNA as template. This piece of cDNA was first cloned into a TA cloning vector PCR 2.1 (Invitrogen, Carlsbad, Calif.). The same cDNA was later subcloned into pCAT3e (Promega) by inserting into the XhoI-SacI site.
- pCAT3e 28h and pCAT3e 28h-1 are mutants of pCAT3e 28b in which ⁇ 51 to ⁇ 22 sequences were deleted and substituted by 15 other nucleotides.
- Jurkat cells were prepared in 2 or 3 T150s at 1:4 or 1:5 dilutions from 80–90% confluent cells. Just before transfection, all cells were pooled in one flask and counted (concentration should be around 40 ⁇ 10 4 per ml.) 11 ⁇ 4 ⁇ 10 6 cells for 10 transfection reactions were spun down in 50 ml conical tubes. Cells were washed 1 ⁇ with half of the original volume of PBS, then resuspended in 44 ml prewarmed fresh Jurkat media (90% RPMI 1640, 10% FBS, 1% L-glutamate, 1% penicillin/streptomycin) so final concentration was 1 ⁇ 10 6 /ml.
- cells were harvested by pipetting the cells from each well to 15 ml conical tubes, making sure to rinse well with cell media so no cells were left behind. They were spun at 2,000 rpms for 5 minutes at room temperature. Media was pipetted off. Each cell pellet was washed 3 ⁇ with 2 ml PBS (PBS was added, vortexed, spun, media pipetted off). As much of the final PBS wash as possible was removed with a pipette tip. 400 ⁇ l of 1 ⁇ Reporter Lysis Buffer (Promega CAT Enzyme Assay System) was added to each cell pipette, and transferred to a 1.5 ml tube. The cell pellet was incubated in lysis buffer at room temp.
- Reporter Lysis Buffer Promega CAT Enzyme Assay System
- FIG. 2 shows the electrophoretic mobility shift analysis of 32P-labeled oligonucleotides preincubated with HeLa cell extract.
- the list of oligos in Table 3 includes two [ FIG. 1A (Seq #4) and ICN 16481 (Seq # 5)] which contain a 12mer motif bearing two sets of G-tetrads separated by 4 nucleotides.
- the motif bearing oligos (lanes 5 and 11 were the only test oligos to give such an oligo-protein shift (Band A) distinct from other phosphorothioate oligos.
- Table 4 compares the inhibitory effect on both mitogen-induced CD28 expression and IL-2 production by certain phosphorothioate oligonucleotides at 5 ⁇ M, with their aptameric ability to form a specific oligo-protein complex when incubated with HeLa nuclear extract, an enriched source of transcription factors.
- FIG. 1A shows that indeed the phosphorothioate G-rich oligos, FIG. 1A and ICN 16481 can act as aptamers in binding to the DNA binding site of Sp1.
- the consequence of this interaction is the prevention of Sp1 binding to the Sp1 site at ⁇ 51 to ⁇ 22 in the promoter region and which inhibits Sp1-mediated transcription of the CD28 gene and decreases expression of the mature CD28 protein.
- ICN 16064 The minimal sequence required for in vitro activity of ICN 16064 was determined by the ability of sequential changes (in bold) to affect ICN 16064-mediated inhibition of CD28 expression in anti-CD3/PMA-activated human T cells and their effect on activated IL-2 production in Jurkat T cells. *Results are expressed relative to the activity of 5 ⁇ M ICN 16064 (100%) whose range of inhibition in seven experiments was 52–79% of CD28 expression and 76–89% of IL-2 production.
Abstract
Description
5′ 3′ | SEQ ID | ||
TTG GAG GGG GTG GTG GGG | FIG. 1A | ||
GGG GAG GAG GGG | ICN | 16481 | |
GGG GTG GTG GGG | ICN 16525 | ||
TTG GAG GGG GAG GAG GGG | ICN 16475 | ||
TTG GAG GGG GAG GTG GGG | ICN 16479 | ||
GGG TTG GAG GGG GTG GTG GGG | ICN 16065 |
While the illustrated sequences are believed to be accurate, the present invention is directed to the correct sequences should errors be found. Oligonucleotides useful in the invention comprise one of these sequences, or part thereof. Thus, it is preferred to employ any of these oligonucleotides as set forth above, or any of the similar oligonucleotides which persons of ordinary skill in the art can prepare from knowledge of the preferred oligonucleotide targets for the modulation of the synthesis of T-cell activation molecules including CD28 and CD28-related molecules. The inhibition or modulation of production of the CD28 and/or CD28 homologues are expected to have significant therapeutic benefits in the treatment of disease. In order to assess the effectiveness of the compositions, an assay or series of assays is required.
TABLE 1 | |||
Seq. No. | | Sequence | |
1 | |
5′ GGG TTC CTC GGG GAG GAG GGG |
|
3′ CCC AAG GAG CCC CTC CTC CCC |
|||
2. | |
5′ GGA GCA |
|
3′ CCT CGT |
|||
3. | 28h-1 | 5′ TCA TCA CAG GGT GCT | |
3′ AGT AGT GTC CCA CGA | |||
4 | ICN 16064 | 5′ TTG GAG GGG GTG GTG GGG | |
5 | ICN 16481 | 5′ GGG GAG GAG GGGCTG GAA | |
6 | ICN 16065 | 5′ GGG TTG GAG GGG GTG GTG GGG | |
7 | ICN 16475 | 5′ TTG GAG GGG GAG GAG GGG | |
8 | ICN 16479 | 5′ TTG GAG GGG GAG GTG GGG | |
9 | ICN 16480 | 5′ TTG GAG GCG GTG GTG GCG | |
10 | ICN 16538 | 5′ TTG GAG CCG GTG GTG GCC | |
11 | ICN 16539 | 5′ TTG GAG GGG CTC CTC GGG | |
12 | ICN 16523 | 5′ TTG GAG CCG GTG GTG G | |
13 | ICN 16525 | 5′ GGG GTG GTG GGG | |
14 | ICN 16526 | 5′ G GGG TTG GGG | |
15 | ICN 16483 | 5′ TG GGG | |
16 | ICN 16482 | 5′ G GGG | |
17 | ICN 16527 | 5′ CAC TGC GGG GAG GGC TGG GG | |
18 | ICN 16528 | 5′ ATG GGG TGC ACA AAC TGG GG | |
19 | ICN 16487 | 5′ AAC GTT GAG GGG CAT | |
20 | ICN 16476 | 5′ TTC CAG CCC CTC CTC CCC | |
21 | ICN 16214 | 5′ AAC CTC CCC CAC CAC CCC | |
22 | SP1 | 5′ ATT CGA TCG GGG CGG GGC GAG C | |
3′ TAA GCT AGC CCC GCC CCG CTC G | |||
23 | AP1 (c-jun) | 5′ CGC TTG ATG AGT CAG CCG GAA | |
3′ GCG AAC TAC TCA GTC GGC CTT | |||
24 | AP2 | 5′ GAT CGA ACT GAC CGC CCG CGG CCC CT | |
3′ CTA GCT TGA CTG GCG GGC GCC GGG GA | |||
25 | NF-KB | 5′ AGT TGA GGG GAC TTT CCC AGG C | |
3′ TCA ACT CCC CTG AAA GGG TCC G | |||
26 | OCT1 | 5′ TGT CGA ATG CAA ATC ACT AGA A | |
3′ ACA GCT TAC GTT TAG TGA TCT T | |||
27 | CREB | 5′ AGA GAT TGC CTG ACG TCA GAG AGC TAG | |
3′ TCT CTA ACG GAC TGC AGT CTC TCG ATC | |||
28 | TFIID | 5′ GCA GAG CAT ATA AGG TGA GGT AGG A | |
3′ CGT CTC GTA TAT TCC ACT CCA TCC T | |||
TABLE 2 |
Identification of oligonucleotide sequence responsible for |
inhibition of CD28 expression and CD28-dependent IL-2 production |
*Relative inhibition | |
of expression |
Oligo ID | Sequence | CD28 | IL-2 |
ICN 16064 | TTG GAG GGG |
100 | 100 |
|
GGG GAG |
100 | 100 |
ICN 16065 | GGG TTG GAG GGG |
100 | 100 |
ICN 16475 | TTG GAG GGG |
100 | 100 |
ICN 16479 | TTG GAG GGG |
100 | 100 |
ICN 16480 | TTG GAG GCGGTG GTG GCG | 31 | 38 |
ICN 16538 | TTG GAG C CG GTG |
40 | 57 |
ICN 16539 | TTG GAG GGG CTC CTC GGG | 44 | 25 |
ICN 16523 | TTG GAG CCG GTG GTG G | 38 | 57 |
ICN 16525 | GGG |
100 | 120 |
ICN 16526 | G GGG TTG GGG | 30 | 39 |
ICN 16483 | |
2 | 2 |
ICN 16482 | |
2 | 2 |
ICN 16527 | CAC TGC GGG GAG GGC TGG GG | 58 | 76 |
ICN 16528 | A TG GGG TGC ACA AAC TGG GG | 51 | 63 |
ICN 16487 | AAC GTT GAG GGG CAT | 26 | 52 |
ICN 16476 | TTC CAG CCC CTC CTC CCC | 29 | 22 |
ICN 16214 | AAC CTC CCC |
4 | 2 |
A 12 mer sequence containing two G quartets separated by four bases confers oligo activity. The minimal sequence required for in vitro activity of ICN 16064 was determined by the ability of sequential changes (in bold) to affect ICN 16064-mediated inhibition of CD28 expression in anti-CD3/PMA-activated human T cells and their effect on activated IL-2 production in Jurkat T cells. | |||
*Results are expressed relative to the activity of 5 μM ICN 16064 (100%) whose range of inhibition in seven experiments was 52–79% of CD28 expression and 76–89% of IL-2 production. |
TABLE 3 |
Nuclear extract protein-binding profiles of |
phosphorothioate oligos (see FIG. 2) |
Oligo | Sequence | Lane No. |
ICN 16064 | TTG GAG GGG |
11, 12 |
|
GGG GAG GAG |
5, 6 |
ICN 16480 | TTG GAG GCG |
7, 8 |
ICN 16538 | TTG GAG CCG |
1, 2 |
ICN 16485 | GTT GGA GAC |
3, 4 |
ICN 16476 | TTC CAG CCC |
9, 10 |
TABLE 4 |
Inhibition of Functional CD28 Expression Correlates With |
the Presence of Specific Oligo-Protein Complex |
Relative inhibition | ||
of expression (%) | Oligo/ |
Oligo | Sequence | CD28 | IL-2 | protein complex |
ICN 16064 | TTG GAG GGG |
100 | 100 | |
ICN | ||||
16481 | GGG GAG GAG |
100 | 100 | Yes |
ICN 16480 | TTG GAG GCG GTG GTG GCG | 31 | 38 | No |
ICN 16538 | TTG GAG CCG |
40 | 57 | No |
ICN 16485 | GTT GGA GAC |
11 | 15 | No |
ICN 16476 | TTC CAG CCC CTC CTC CCC | 29 | 22 | No |
ICN 16214 | AAC CTC CCC |
4 | 2 | No |
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US09/331,204 US6994959B1 (en) | 1996-12-27 | 1997-12-19 | G-rich oligo aptamers and methods of modulating an immune response |
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CA (1) | CA2278031A1 (en) |
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IL (1) | IL130192A0 (en) |
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CZ227199A3 (en) | 1999-12-15 |
NO993170D0 (en) | 1999-06-25 |
EP0968226A1 (en) | 2000-01-05 |
JP2002512599A (en) | 2002-04-23 |
KR20000069658A (en) | 2000-11-25 |
SK84599A3 (en) | 2000-07-11 |
WO1998029430A1 (en) | 1998-07-09 |
NO993170L (en) | 1999-08-25 |
IL130192A0 (en) | 2000-06-01 |
SI20117A (en) | 2000-06-30 |
BR9714438A (en) | 2000-03-21 |
PL334197A1 (en) | 2000-02-14 |
HU0000769A (en) | 2000-07-28 |
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